Detection of Molecule-Nanoparticle Interactions with Ligand Shells

ABSTRACT

A quartz crystal microbalance coated with functionalized nanoparticles used to detect molecule-nanoparticle interactions to assist with characterization of difficult to predict molecule-nanoparticle interactions for novel ligand chemistries and, particularly, mixed ligand nanoparticles exhibiting different ligand morphologies, in order to quantify nanoparticle-molecule interactions independently from more complex solvation requirements.

BACKGROUND OF THE INVENTION 1) Field of the Invention

The present invention relates to a quartz crystal microbalance (QCM)approach to more broadly quantify molecule-NP interactions via vaporphase uptake into solid NP-films independent from solvation constraints.

2) Description of Related Art

Nanoparticles (NPs) have gained widespread interest for a wide array ofapplications such as chemical and biological sensing, drug delivery fornanomedicine, self-assembly, and removal of contaminants. Theperformance of a NP for an application is largely influenced by itsintermolecular interactions with the local environment as determined bythe character of the ligand shell. The ligand shell is the ultimateinterface of the NP with the outside world and thus governs interactionswith other objects. The properties affected by the ligand shell thusrange from solubility, to self-assembly, drug delivery,biocompatibility, and targeted molecular uptake. Mixtures of ligandshave been shown to enable hybrid behaviors, e.g., NPs with extensivehydrophobic or fluorine content can exhibit solubility in water andother aqueous media.

The morphology of mixed ligand shells also significantly modifies NPbehavior. On flat substrates, ligand mixtures phase separate to reducethe enthalpic interfacial area where the surface tension has a monotonicdependence on the ensemble composition. Here, the nearest-neighbormolecular environment within each phase is identical to the mono-ligandfilm.

In other words, ligands phase separated over sufficient distance willinteract independently with the external environment. Janus NPs areanalogously phase separated with ligand domains on opposite sides ofeach NP. Janus NPs thus exhibit a monotonic continuum of behaviorprincipally corresponding to the ensemble of two mono-ligandenvironments. Due to high curvature, mixed ligand NPs can also exhibitpatchy and stripe-like ligand morphologies when coupled with appropriatepairs of ligands having different length. The lowest free-energyconfiguration can promote mixed ligand interfaces to increaseconformational entropy of the longer ligand. Here the longer ligandsexplore additional conformational space when proximal to the shorterligands.

This remarkable entropy-driven ordering is widely documented to occurunder specific conditions. Patchy and stripe-like ligand morphologieshave small <2 nm ligand domains that are dominated by the mixed-ligandinterface. Patchy and stripe-like NPs can thus exhibit non-monotonictrends in behavior where the local molecular environment behavesdistinctly from the bulk ensemble. This follows naturally with theincreasing contribution of dissimilar nearest-neighbor ligands wheretheir interface behaves differently from either ligand alone. Thechanges in molecule-NP interactions are not yet predictable a priori andare tedious to measure where each molecule-NP interaction is testedindividually, typically with a solubility limit measurement. Thenon-monotonic behavior exhibited by patchy and stripe-like nanoparticleshas been explained by a combination of cavitation suppressingselective-solvent uptake or by confinement enhancing solvent uptake intoappropriately matched molecular environments. Cavitation and confinementthus work in opposing directions where the balance between the two leadsto variable non-monotonic molecule-NP behaviors. For example, a recentreport with mixed ligand amphiphilic NPs having 67% hydrophobic ligandwere most soluble in polar alcohols and this alcohol solubility wasreduced when increasing the hydrophilic ligand content.

NP saturation experiments with different solvents or solvationconditions are widely used to quantify solvent-NP interactions. Incontrast, more general measurements of molecule-NP interactions do notnecessarily require a solvation shell. For example, NP drug loading is aseparate criterion from solvation in the delivery medium.

Accordingly, it is an object of the present invention to provide amethod for detecting the interaction of molecules with nanoparticles ofdiverse surface chemistries in order to assist with characterization ofdifficult to predict molecule-nanoparticle interactions for novel ligandchemistries and, particularly, mixed ligand nanoparticles exhibitingdifferent ligand morphologies, in order to quantifynanoparticle-molecule interactions independently from more complexsolvation requirements.

SUMMARY OF THE INVENTION

The above objectives are accomplished according to the present inventionby providing in a first embodiment a quartz crystal microbalance methodfor detecting molecular interactions with nanoparticles to predictmolecule-nanoparticle interactions comprising. The method may includepreparing a nanoparticle film on a quartz crystal, exposing thenanoparticle film to at least one molecular vapor; and quantifying massuptake via analyzing a resonant frequency of the quartz crystal.Further, the method may include correlating nonmonotonic uptake trendsto ligand shell morphologies as a function of confinement and cavitationeffects. Still further, non-solvents may be used to quantifymolecular/nanoparticle interactions. Yet still, determining morphologyof at least one molecule/nanoparticle shell may be accomplished viaanalyzing nuclear magnetic resonance chemical shifts. Again, interfacesof different ligands have a different chemical shift wherein extent ofchemical shift may be a weighted average of at least one local ligandenvironment. Still again, molecular uptake may be measured withoutrequiring a solvation sphere. Further again, the nanoparticle film maybe formed by spin coating. Still yet, the method may include comparingmolecular mass uptake to nanoparticle film mass to quantify extent ofuptake. Again, the method may include probing for non-monotonic trendsin molecule-nanoparticle interactions with changes to ligandcomposition. Still further, the method may include eliminatingnanoparticle size distribution as a variable via employing ligandexchange.

In an alternative embodiment, the current disclosure provides a methodto quantify mixed ligand shell molecule-nanoparticle interaction. Themethod may include measuring vapor phase uptake of molecules into asolid nanoparticle film, deposited on a crystal, via nuclear magneticresonance, wherein the nanoparticle film comprises mixed ligandnanoparticles with constant size and variable composition; and themethod is independent of solvation criteria. Further, the crystal may bequartz. Still further, patchy ligand morphologies may exhibit moremolecule uptake than either stripe-like or mono-ligand nanoparticles.Yet again, measurements may be taken without requiring a solvationshell. Further still, ligand stripping may be employed. Yet further, themethod may include eliminating nanoparticle size distribution as avariable via employing ligand exchange.

BRIEF DESCRIPTION OF THE DRAWINGS

The construction designed to carry out the invention will hereinafter bedescribed, together with other features thereof. The invention will bemore readily understood from a reading of the following specificationand by reference to the accompanying drawings forming a part thereof,wherein an example of the invention is shown and wherein:

FIG. 1 shows NPs with mixed ligand shells can have variable compositionand ligand morphology.

FIG. 2 illustrates Porod plots of aminated, 0F, 25F, 52F, 100F NPsolutions.

FIG. 3 shows NMR of the ligated nanoparticles after washing showing theabsence of free ligand.

FIG. 4 shows NP bound ligand composition measured by NMR after ligandstripping with iodine, resulting in the corresponding disulfide mixtures

FIG. 5 shows correlation of ligand exchange solution composition to thecomposition of bound ligands on nanoparticle surfaces (DDT and PFOTligands).

FIG. 6 shows trends of ¹⁹F NMR chemical shift for —CF3 (a) and the 7th—CF2- (b) on PFOT as a function of NP ligand composition (DDT/PFOT).

FIG. 7 shows: (a) Scheme of QCM setup with controlled solvent vapor; (b)A characteristic vapor response curve for the 0F NP film with1,4-difluorobenzene where the shaded region represented the solventuptake; (c) A sequential series of solvent measurements for a 0F NP filmwith benzene, 1,4-difluorobenzene, 1,3,5-trifluorobenzene,1,2,4,5-tetrafluorobenzene, and hexafluorobenzene vapor, respectively.

FIG. 8 shows mass uptake of benzene vapor in NP films as a function ofPFOT:DDT mixed ligand shell composition.

FIG. 9 shows mass uptake of solvent vapors in NP films as a function ofPFOT:DDT mixed ligand shell composition: (a) 1,4-difluorobenzene; (b)1,3,5-trifluorobenzene; (c) 1,2,4,5-tetrafluorobenzene; and (d). Guidelines are indicated with dashes.

FIG. 10 shows a comparison of different benzene derivative uptakes intothe 0F and 100F NP films.

FIG. 11 shows table S1 displaying NP diameter and distribution resultsfrom Porod fitting results.

FIG. 12 shows table S2 displaying ligand shell compositions and surfacedensities for mixed nanoparticles.

FIG. 13 shows QCM solvent series of the 20F NPs, the asterisk representsthe solvent line coming undone during the experiment.

FIG. 14 shows QCM solvent series of the 31F NPs.

FIG. 15 shows QCM solvent series of the 39F NPs.

FIG. 16 shows QCM solvent series of the 52F NPs.

FIG. 17 QCM solvent series of the 39F NPs. For this series 1.8tau wasmet and when fit the films were nearly equilibrated. The fitted valueswere used to calculate the relative uptake.

FIG. 18 shows QCM solvent series of the 73F NPs.

FIG. 19 shows QCM series of the 93F NPs.

FIG. 20 shows QCM series of the 100F NPs.

FIG. 21 shows ¹⁹F NMR shift results for the —CF₃ and 7th CF₂ unit of thePFOT ligand.

It will be understood by those skilled in the art that one or moreaspects of this invention can meet certain objectives, while one or moreother aspects can meet certain other objectives. Each objective may notapply equally, in all its respects, to every aspect of this invention.As such, the preceding objects can be viewed in the alternative withrespect to any one aspect of this invention. These and other objects andfeatures of the invention will become more fully apparent when thefollowing detailed description is read in conjunction with theaccompanying figures and examples. However, it is to be understood thatboth the foregoing summary of the invention and the following detaileddescription are of a preferred embodiment and not restrictive of theinvention or other alternate embodiments of the invention. Inparticular, while the invention is described herein with reference to anumber of specific embodiments, it will be appreciated that thedescription is illustrative of the invention and is not constructed aslimiting of the invention. Various modifications and applications mayoccur to those who are skilled in the art, without departing from thespirit and the scope of the invention, as described by the appendedclaims Likewise, other objects, features, benefits and advantages of thepresent invention will be apparent from this summary and certainembodiments described below, and will be readily apparent to thoseskilled in the art. Such objects, features, benefits and advantages willbe apparent from the above in conjunction with the accompanyingexamples, data, figures and all reasonable inferences to be drawntherefrom, alone or with consideration of the references incorporatedherein.

DETAILED DESCRIPTION OF A PREFERRED EMBODIMENT

With reference to the drawings, the invention will now be described inmore detail. Unless defined otherwise, all technical and scientificterms used herein have the same meaning as commonly understood to one ofordinary skill in the art to which the presently disclosed subjectmatter belongs. Although any methods, devices, and materials similar orequivalent to those described herein can be used in the practice ortesting of the presently disclosed subject matter, representativemethods, devices, and materials are herein described.

Unless specifically stated, terms and phrases used in this document, andvariations thereof, unless otherwise expressly stated, should beconstrued as open ended as opposed to limiting. Likewise, a group ofitems linked with the conjunction “and” should not be read as requiringthat each and every one of those items be present in the grouping, butrather should be read as “and/or” unless expressly stated otherwise.Similarly, a group of items linked with the conjunction “or” should notbe read as requiring mutual exclusivity among that group, but rathershould also be read as “and/or” unless expressly stated otherwise.

Furthermore, although items, elements or components of the disclosuremay be described or claimed in the singular, the plural is contemplatedto be within the scope thereof unless limitation to the singular isexplicitly stated. The presence of broadening words and phrases such as“one or more,” “at least,” “but not limited to” or other like phrases insome instances shall not be read to mean that the narrower case isintended or required in instances where such broadening phrases may beabsent.

The current disclosure provides a quartz crystal microbalance (QCM)approach to more broadly quantify molecule-NP interactions via vaporphase uptake into solid NP-films independent from solvation constraints.The composition and morphology of mixed ligand shells were found toexhibit pronounced non-monotonic behavior that deviated from continuumthermodynamics, highlighting the influence of ligand morphology uponabsorption/adsorption. Alkyl and perfluorinated thiols were used as amodel case with constant core-size distribution. The ligand morphologywas determined by 19F NMR. Molecule uptake into NPs was measured withfive benzene derivatives with varied degree of fluorination. For thecases examined, QCM measurements revealed enhanced uptake for patchymorphologies and suppressed uptake for stripe-like morphologies. Theseresults contrast with insights from solubility measurements alone whereQCM sometimes identified significant molecular uptake with poorsolvents. This QCM method thus provides new insights to molecule-NPinteractions independent of the solvation shell.

The current disclosure has developed a quartz crystal microbalance (QCM)method to quantify molecule-NP interactions via vapor phase uptake intosolid NP thin films. QCM has previously been used on NP films to monitorchemiresistence, detect various biomaterials, and to analyze cellularinteractions due to its high sensitivity. The approach uses miniscule NPquantities and can uniquely quantify molecule-NP interactions withnon-solvents. In one embodiment, the current disclosure examines a modelsystem consisting of 1.8 nm gold NPs with a variable combination ofshort fluorophilic ligands and long lipophilic ligands that wereexpected to form patchy and stripe-like ligand morphologies. Themolecule-NP interactions were examined for a systematic series offluorinated benzene derivatives as a function of NP ligand compositionand morphology. Nonmonotonic trends in solvent uptake were correlated tothe ligand shell morphologies as a function of confinement andcavitation effects.

Results and Discussion: Preparation of Mixed-Ligand NP

A range of mixed ligand NPs were synthesized under conditions expectedto form patchy and stripe-like ligand morphologies. The generalrequirements for these morphologies are a combination of ligands withdifferent lengths on a NP of suitable curvature, e.g. generally ˜2-8 nmin diameter. A recent experimental and computational study examinedmixtures of fluorophilic and lipophilic ligands on 2-4 nm gold NPs wherethe length of the ligands were varied across a wide composition range todetermine the impact on ligand morphology. Janus regions were observedif the ligands had similar length. The flexible lipophilic ligandsneeded to be >4-6 carbons longer than the stiff fluorinated ligands toform patchy or stripe-like morphologies. The morphologies were mappedfor patchy (0-30 mol % fluorinated and 60-100 mol % fluorinated) andstripe-like (30-60 mol % fluorinated) morphologies. Prior work suggeststhat the current disclosure's selection of DDT and PFOT (4 carbondifference) with 1.8 nm diameter Au NPs will yield patchy andstripe-like ligand morphologies.

Mixed ligand NPs were prepared using standard methods. See, Hostetler,M. J.; Green, S. J.; Stokes, J. J.; Murray, R. W. Monolayers in ThreeDimensions: Synthesis and Electrochemistry of ω-FunctionalizedAlkanethiolate-Stabilized Gold Cluster Compounds. J. Am. Chem. Soc.1996, 118, 4212-4213, which is hereby incorporated by reference. Murrayet. al developed a method for mixed ligand NPs using a post-synthesisligand exchange. This process was later expanded to displace weaklybound amine or phosphorous ligands with stronger binding thiol ligands.See Brown, L. O.; Hutchison, J. E. Convenient Preparation of Stable,Narrow-Dispersity, Gold Nanocrystals by Ligand Exchange Reactions. J.Am. Chem. Soc. 1997, 119, 12384-12385, which is hereby incorporated byreference. The NP core size is thus constant and is decoupled from thefinal ligand chemistry, see FIG. 1. FIG. 1 shows that NPs with mixedligand shells can have variable composition and ligand morphology.Displacement of weakly bound ligands (R1) with strong binding ligands(R2, R3) yields systematic NP series with constant NP core sizedistribution and variable ligand shells.

Our synthesis used a procedure for aminated <5 nm Au NPs followed byamine displacement with lipophilic DDT and fluorophilic PFOT ligands.See Jana, N. R.; Peng, X. Single-Phase and Gram-Scale Routes towardNearly Monodisperse Au and Other Noble Metal Nanocrystals. J. Am. Chem.Soc. 2003, 125, 14280-14281, which is hereby incorporated by reference.The NP behavior followed broad expectations where PFOT-rich NPsprecipitated from toluene whereas DDT-rich NPs were toluene soluble.SAXS was used to confirm the NP size distributions. Comparison of theAm. NPs to ligand displaced NPs resulted in similar scattering curveswith the nearly identical q-positions for local minima and maxima, seeFIG. 2. (FIG. 2 illustrates Porod plots of aminated, 0F, 25F, 52F, 100FNP solutions. Data points and best-fit lines are indicated. Scatteringdata are offset vertically for clarity.) Each dataset was well fittedusing a hard sphere form factor with a Gaussian size distribution. Theresults indicated 1.6-2.0±0.4-0.6 nm NP diameter distribution, with someminor differences between the converged fits (see FIG. 11, Table S1).Thus, ligand displacement was shown to not significantly alter the NPcore size distribution.

The resulting mixed ligand NPs were rigorously purified beforedetermination of the ligand surface density and composition. Thesynthesis solution contained a surfactant to improve NP solubilityhowever residual surfactant would influence subsequent measurements ofmolecule-NP interactions. The NPs were thus purified with iterativedispersal/precipitation cycles. The solubility of the NPs changedmarkedly with the cleaning steps as well documented before in Dass, A.;Guo, R.; Tracy, J. B.; Balasubramanian, R.; Douglas, A. D.; Murray, R.W. Gold Nanoparticles with Perfluorothiolate Ligands. Langmuir 2008, 24,310-315 and Yonezawa, T.; Onoue, S. Y.; Kimizuka, N. Self-OrganizedSuperstructures of Fluorocarbon-Stabilized Silver Nanoparticles. Adv.Mater. 2001, 13, 140-142, both of which are hereby incorporated byreference. NMR spectra after six wash cycles were without sharp peaksassociated with free-ligand or surfactant, see FIG. 3. (FIG. 3 providesNMR of the ligated nanoparticles after washing showing the absence offree ligand. NMR of the DDT thiol is represented by the red a while thefree PFOT is denoted at the bottom of the image. *TFT added forsolubility. The NPs were named by the mol % of PFOT in the DT/PFOTligand shells, see FIG. 4.) An aliquot of the mixed ligand NPs was thenstriped of ligands using metallic iodine to improve quantification ofthe formerly-bound ligand population, see FIG. 4. (FIG. 4 shows the NPbound ligand composition measured by NMR after ligand stripping withiodine, resulting in the corresponding disulfide mixtures.) The ratio ofbound ligands was fully tunable from 0-100 mol % PFOT and with smalldeviation from the exchange solution, see FIG. 5 and FIG. 12 showingTable S2, which shows ligand shell compositions and surface densitiesfor mixed nanoparticles. (FIG. 5 shows correlation of ligand exchangesolution composition to the composition of bound ligands on nanoparticlesurfaces (DDT and PFOT ligands)). A non-convolved internal standard(1,4-difluorobenzene) was included in the same ligand strippingexperiments to determine the ligand surface concentration. A fringebenefit of the chosen internal standard is that it also improves NPsolubility. Comparison of the ligand concentration to the NPconcentration determined by optical absorption experiments yielded thesurface ligand density. The ligand surface density for all mixed-ligandNPs examined were within the range of 1-5 #/nm², consistent with similarreports of NPs without detectable free-ligand, see FIG. 12, Table S2). Aseries of purified DDT/PFOT mixed ligand NPs were thus prepared withconstant size distribution.

Determination of Ligand Morphology by ¹⁹F NMR

Numerous methods can determine the morphology of mixed ligand NP shells.Common methods include NMR, mass spectroscopy, Scanning TunnelingMicroscopy, MALDI-TOF, UV-Vis paired with Cryo-TEM, Electron SpinResonance, Infrared Spectroscopy paired with STM, and contact anglemeasurements. The current disclosure used the method developed byPasquato et. al to determine the mixed ligand morphology using trends in¹⁹F NMR chemical shifts. See Sologan, M.; Marson, D.; Polizzi, S.;Pengo, P.; Boccardo, S.; Pricl, S.; Posocco, P.; Pasquato, L. Patchy andJanus Nanoparticles by Self-Organization of Mixtures of Fluorinated andHydrogenated Alkanethiolates on the Surface of a Gold Core. ACS Nano2016, 10, 9316-9325, which is hereby incorporated by reference. Themethod was demonstrated with similar fluorophilic/lipophilic ligandmixtures and was supported by computational predictions.

By using ¹⁹F NMR, which has a high sensitivity due to the large chemicalshift range, small changes in the local environment result in largerchemical shifts. If a ligand is surrounded by identical neighbors,similar to a mono-ligand film, then the chemical shift is insensitive tocomposition changes. Interfaces of different ligands have a differentchemical shift where the extent of the shift is a weighted average ofthe local ligand environments. Distinct trends in chemical shift withligand composition are anticipated for different sequences of ligandmorphologies. Linear decays, exponential decays, and sigmoidal decayswere previously correlated to random packing, patchy/Janus, andpatchy/stripe-like morphologies, respectively.

The ¹⁹F NMR measurements of both the CF₃ group centered near ˜80 ppm andthe CF₂ group centered near ˜127 ppm both exhibited sigmoidal trends inchemical shift with ligand composition for the synthesized NP series,see FIG. 6. (FIG. 6 shows trends of ¹⁹F NMR chemical shift for —CF₃ (a)and the 7th —CF₂- (b) on PFOT as a function of NP ligand composition(DDT/PFOT). A sigmoidal guide line is presented. Interpreted transitionsin ligand morphology are indicated with dashed drop lines.). Both curvesexhibit similar trends in the chemical shift decay suggesting transitionfrom patchy to stripe-like to patchy morphologies, similar to analogousNP preparations.

QCM Quantification of Molecule-NP Interactions

A custom QCM apparatus was constructed to quantify molecule-NPinteractions. Each NP-film was prepared directly on a quartz crystal byspin coating NP solutions. The NP-film was subsequently exposed tosolvent vapor and the mass uptake was quantified by the shiftingresonant frequency of the quartz crystal. An advantage of QCM is rapidreal time feedback with high mass-resolution, the use of minute NPquantities, and the ability to measure molecule uptake without requiringa solvation sphere. Early experiments guided selection of films thatwere approximately 60 nm thick or less to minimize diffusion time. Thisthickness was consistently achieved by using a 1 wt % NP solution and aspin speed of 5,000 rpm for an even thin film on the crystal surface.Slower spin speeds (<2,000 rpm) resulted in >100 nm thick films withexcessive equilibration times. For typical experiments, the frequencyresponse to vapor was exponential with a time constant that ranged foreach film from 8-14 minutes.

A typical uptake experiment is shown in FIG. 7 b. Comparison of themolecule mass uptake to that of the NP-film thus quantifies the relativeextent of uptake. The experiment is easily extendable by examiningmultiple molecule vapors sequentially, see FIG. 7 c. (FIG. 7 shows: (a)scheme of QCM setup with controlled solvent vapor; (b) a characteristicvapor response curve for the 0F NP film with 1,4-Difluorobenzene wherethe shaded region represented the solvent uptake; (c) a sequentialseries of solvent measurements for a 0F NP film with benzene,1,4-difluorobenzene, 1,3,5-trifluorobenzene, 1,2,4,5-tetrafluorobenzene,and hexafluorobenzene vapor, respectively.) Typical experiments yielded10-35% molecule mass uptake relative to the film mass. The completevapor series for each particle composition can be found in thesupplementary information, see FIGS. 13-20. FIG. 13 shows QCM solventseries of the 20F NPs, the asterisk represents the solvent line comingundone during the experiment. FIG. 14 shows QCM solvent series of the31F NPs. FIG. 15 shows QCM solvent series of the 39F NPs. For thisseries 1.8tau was met and when fit the films were nearly equilibrated.The fitted values were used to calculate the relative uptake. FIG. 16shows QCM solvent series of the 52F NPs. FIG. 17 shows QCM solventseries of the 39F NPs. For this series 1.8tau was met and when fit thefilms were nearly equilibrated. The fitted values were used to calculatethe relative uptake. FIG. 18 shows QCM solvent series of the 73F NPs.FIG. 19 shows QCM series of the 93F NPs. FIG. 20 shows QCM series of the100F NPs. The asterisk denotes the solvent line coming undone. Theeffect of ligand morphology on molecular uptake are presented next bycomparison of the QCM response of NPs with different mixed ligandcompositions.

Correlation of Ligand Morphology to molecule-NP Interactions

The simplest approach for series comparisons of molecule-NP interactionsis with variable NP and constant molecule vapor. This eliminates theneed to quantify and vary the vapor pressure for direct moleculecomparisons. Slight variations in composition of mixed ligand NPs cantranslate into significant macroscopic effects where NPs can become moresoluble in a perceived non-solvent or vice versa. Such non-monotonicbehavior is typically quantified for NPs with solution saturationexperiments. A distinct benefit of the current disclosure's QCM methodis the quantification of molecule-NP interactions for non-solvents. Thesystematic series of mixed ligand NPs prepared above are idealcandidates for the development and testing of this new QCM basedapproach to probe for non-monotonic trends in molecule-NP interactionswith changes to the ligand composition and thus ligand morphology. Thecurrent disclosure's synthesis strategy notably eliminates thenanoparticle size distribution as a variable by using a ligand exchangestrategy. Recent experimental and computational work coupled with thecurrent disclosure's ¹⁹F NMR measurements suggest a sequence of patchyand stripe-like morphologies here.

First, the uptake of benzene vapor was systematically examined using arange of NP surface compositions, see FIG. 8, which shows mass uptake ofbenzene vapor in NP films as a function of PFOT:DDT mixed ligand shellcomposition. NPs with only DDT ligands uptook 15 wt % benzene mass andNPs with only PFOT ligands uptook 11% benzene mass. These two extremepoints constrain the possible trajectories for monotonic behavior trendsto be in intermediate to these two values. The NPs used here withstripe-like morphologies (39-59 mol % PFOT) exhibited reduced solventuptake relative to the two mono-ligand cases, indicative of molecularcavitation. In contrast, the NPs used here with patchy morphologies(both PFOT-poor and PFOT-rich) exhibited markedly enhanced uptake,indicative of molecular confinement. For example, the 20F NPs up took 28wt % benzene, a ˜2× increase relative to the 0F NPs despite the additionof a fluorophile.

Clearly, the molecule-NP interaction is sensitive to the character ofthe ligand morphology. The current disclosure notes that ¹⁹F NMR of 31Fwas at a transition between patchy and stripe-like morphologies and wasthus excluded from discussion of generalized trends due to ambiguity.The trends in uptake may be attributed to the nominal dimension of theligand domains, increasing when transitioning from stripe-like to patchymorphologies. Molecular confinement, e.g., within the gaps between theshort and tall ligands, requires that the ligand domains accommodateboth the molecule functionality and dimensions. This was rationalizedwith a confinement argument in a prior study, Rycroft, C. H.;Barenblatt, G. I.; Yamagata, K.; Kondo, T.; Hayashi, S.; Shitamukai, A.;Konno, D.; Matsuzaki, F.; Onami, S.; Nakayama, H.; et al. The Role ofNanostructure in the Wetting Behavior of Mixed-Monolayer-Protected MetalNanoparticles. Proc. Natl. Acad. Sci. U. S. A. 2008, 110, 6240-6240,which is hereby incorporated by reference, where adding 14 mol % of asolvophobic ligand led to a 30% increased saturation concentration ascompared to the mono-ligand NP.

An interesting feature is that even the 100F NPs with mono-ligand PFOTuptook 11 wt % benzene; this interaction of benzene would be missed bysolubility measurements alone as the 100F NPs are nearly insoluble inbenzene. This marked difference between solubility measurements and QCMsolvent uptake exhibit the distinction between molecular uptake and thecapability to form a favorable solvation shell. Thus, QCM enablesadditional insights to quantify molecule-NP interactions independent ofsolubility.

Next a systematic series of benzene derivatives were examined withvariable extent of fluorination to determine the effect on overallmolecule-NP interactions. The derivatives included 1,4-difluorobenzene,1,3,5-trifluorobenzene, 1,2,4,5-tetrafluorobenzene, andhexafluorobenzene and were deliberately selected without permanentmolecular dipoles. Each solvent was examined across the same series ofNP compositions and morphologies as above with benzene, see FIG. 9 andFIG. 21. Analogous behavior to benzene was found in all cases where 1)relative to the mono-ligand cases, the patchy NPs exhibited enhanceduptake corresponding to confinement effects and 2) relative to themono-ligand cases, the stripe-like NPs exhibited reduced solvent uptakecorresponding to cavitation effects. These generalized behaviors for thearomatic molecule series suggests an important role of the relativelyconstant molecular shape, size, and presence of the aromatic ring. Asexpected with a like-dissolves-like argument, the 0F 100F NPs exhibitedmore uptake for highly fluorinated benzene derivatives (low Hildebrandparameter), and vice versa, see FIG. 10. The favorable interaction ofPFOT with fluorinated benzene derivatives may be due to either a reduceddifference in relative polarizability (dispersion forces) or possiblythe presence of weak halogen bonding. Other studies have shown thatweakly attractive interactions exist between fluorinated alkanes andelectron deficient aromatics. See, Kawahara, S. I.; Tsuzuki, S.;Uchimaru, T. Theoretical Study of the C—F/π Interaction: AttractiveInteraction between Fluorinated Alkane and an Electron-Deficientπ-System. J. Phys. Chem. A 2004, 108, 6744-6749, and Cavallo, G.;Metrangolo, P.; Milani, R.; Pilati, T.; Priimagi, A.; Resnati, G.;Terraneo, G. The Halogen Bond. Chem. Rev. 2016, 116, 2478-2601, both ofwhich are hereby incorporated by reference.

QCM thus quantifies non-monotonic trends for molecule-NP interactionsthat are influenced by possible contributions from size/shape, ligandmorphology, and chemical nature. The ability to measure these behaviorsis crucial to support further investigation, both experimental andcomputational, into the molecular mechanisms.

Conclusion

A method to quantify mixed ligand shell molecule-NP interactions wasdeveloped that is independent of solvation criteria. A QCM apparatus wasused to measure the vapor phase uptake of molecules into solid NP thinfilms. A series of mixed ligand NPs with constant size and variablePFOT/DDT composition were prepared and were confirmed by ¹⁹F NMR to havea range of ligand shell morphologies. The NPs uptake was measured with asystematic series of fluorinated benzene derivatives. The relative massuptake was non-monotonic with NP ligand shell composition in all cases.For the cases examined, patchy ligand morphologies were found to exhibitmore molecule uptake than either stripe-like or the analogousmono-ligand NPs. This enhanced uptake was attributed to confinementeffects. In contrast, stripe-like morphologies exhibited decreasedmolecule uptake relative to the mono-ligand NPs, consistent withcavitation effects. These results highlight the role of ligand shellmorphology on molecule-NP interactions. Notably the technique enabledmeasurements with non-solvents. The ability to measure interactionswithout a solvation shell leads to a more complete understanding ofmolecule-NP interactions.

Experimental Methods: Materials

Gold trichloride (99.9%) was obtained from Strem Chemical and storedunder inert atmosphere. α,α,α-Trifluorotoluene (≥99%, TFT) and anhydrousiodine lumps (99.99%, under argon) were obtained from BeanTown Chemical.Tetrabutylammonium borohydride (≥98%) and didodecyldimethylammoniumborohydride (≥98%) were purchased from TCI America and stored underargon atmosphere before use. Potassium thioacetate (98%), benzene (99%),and 1-dodecane thiol (98%, DDT) were obtained from Alfa Aesar and usedas received. 1H, 1H, 2H, 2H-Perfluoro-1-iodooctane iodide (>95%) and1,3,5-trifluorobenzene (97%) were obtained from Matrix Scientific andused as received. Hexafluorobenzene (97%), 1,2,4,5-tetrafluorobenzene,and 1,4-difluorobenzene were obtained from Oakwood Chemical and used asreceived. Chloroform-D (99.8%) and benzene-D6 were purchased fromCambridge Isotope Laboratories Inc. and used as received. Toluene(≥99.5%) obtained from Fisher Chemical was subjected to four cycles offreeze-pump-thaw and dried over molecular sieves prior to use.

1H,1H,2H,2H-Perfluoro-1-octanethiol (PFOT) Synthesis

In a round bottom flask, potassium thioacetate was combined with2-(perfluorohexyl)ethyl iodide in a 1.1:1 molar ratio along with THF. Acondenser was connected to the flask and the reaction vessel was sealedand subjected to three cycles of freeze-pump-thaw to remove excessoxygen. It was then filled with inert nitrogen gas and heated for 5 hrat 50° C. The product was collected through filtration and the excessTHF was removed by evaporation. The crude 1H,1H,2H,2H-perfluorooctylthioacetate was purified through vacuum distillation at 70° C., purityand structure was verified with 1H NMR spectroscopy. To obtain thedeprotected thiol the purified thioacetate was added to a flask chargedwith 90 mL of ethanol and 40 mL of concentrated hydrochloric acid. Acondenser was connected to the flask and the reaction vessel was sealedand subjected to three cycles of freeze-pump-thaw to remove dissolvedoxygen. The vessel was filled with inert nitrogen gas and the reactionwas heated for 13 hours at 90° C. The crude thiol was extracted threetimes with 100 mL of hexanes and washed with 100 mL of deionized waterand then dried overnight with magnesium sulfate. The magnesium sulfatewas removed through filtration and the excess hexanes was removedthrough evaporation before the crude thiol was purified through vacuumdistillation. The final purity and structure was verified using ¹H NMRspectroscopy.

Amine-Stabilized NP (Am. NP) Synthesis

In an inert argon glovebox atmosphere, 90 mg of gold(III) chloride wascombined with a 0.1 M didodecyldimethylammonium bromide (DDAB) intoluene surfactant solution in a 125 mL Erlenmeyer flask. The solutionwas gently stirred until the precursor dissolved turning the solution adark orange color. To this solution 216 μL of dodecylamine was addedwhile stirring, it was then stirred until the dark orange color turnedto a light-yellow. In a separate vessel 300 mg of tetra-n-butylammoniumbromide (TBAB) was dissolved in 12 mL of the 0.1 M DDAB stock solution,the TBAB solution was then placed in a syringe. Both the flask andsyringe were sealed under argon and taken out of the inert atmosphere.The gold precursor solution was then stirred at 1,500 rpm. Once thesolution reached 1,500 rpm the TBAB solution was injected into thestirring flask, it immediately changed from a light-yellow color to adeep red. The resulting Am. NP solution was immediately used for liganddisplacement.

Ligand Displacement Procedure

A premade ligand solution containing the desired ratio of DDT/PFOT wasinjected/added to the Am. NPs immediately after synthesis. For mixedligand NPs, the premade ligand solution was kept at a 1:1 totalthiol:gold molar ratio and the proportion of each ligand in the solutionwas adjusted based on the desired shell composition. Post injection, theAuNPs were stirred for fifteen minutes at room temperature and thenboiled at 120° C. for 20 min for the thiols to displace the dodecylamineligands. Post boiling, the NPs were immediately washed six times usingfour toluene washes and two α,α,α-trifluorotoluene (TFT) washes toremove excess surfactant and excess ligands. After the washing cycleswere complete, the particles were collected by centrifugation frommethanol and stored as a powder. The resulting NP batches were termed xFaccording to the final ligand shell composition, vide infra, where theNPs had x mol % PFOT.

NMR Experiments

¹H NMR experiments were carried out on a Bruker Avance III-HD 300 MHz.¹⁹F experiments were carried out on a Bruker Avance III-HD 400A MHz NMR.The ¹H chemical shifts are referenced to deuterated chloroform, while¹⁹F chemical shifts are referenced to TFT. An external reference ofCFCl₃ was used to shift-correct the ¹⁹F spectra to ensure correct peakpositioning.

NP purity was analyzed using ¹H NMR to determine the presence of excesssurfactant and unreacted ligand. A typical procedure involved dissolving5 mg of NPs in either deuterated chloroform or TFT for the heavilyfluorinated particles using ultrasonic agitation. The composition of NPligand shell was measured after ligand stripping using I₂ decomposition.Here, 5 mg of NPs were dissolved in deuterated chloroform before 1-3 mgof metallic iodine was added. The solution was gently mixed at 250 rpmusing a shaker until complete dissolution of the iodine occurred. It wasthen allowed to sit overnight to ensure complete disulfide formation.The black precipitate and iodine was removed, and the disulfides weremeasured using 128 scans on the 1H NMR. The ligand morphology wasdetermined using ¹⁹F NMR measurements with 5 mg of NPs were dissolved ina mixture of TFT/C₆D₆ (97/3 wt %). The particles were dispersed with abath sonicator and were scanned using a 100 ppm window centered at −100ppm with 256 scans.

Small-Angle X-ray Scattering (SAXS)

X-ray experiments were conducted using a SAXSLAB Ganesha at the SouthCarolina SAXS Collaborative. A Xenocs GeniX3D microfocus source was usedwith a Cu target to generate a monochromated beam with a 0.154 nmwavelength. The instrument was calibrated using National Institute ofStandards and Technology (NIST) reference material 640 c silicon powderwith the peak position at 2θ 28.44 where 2θ is the total scatteringangle. A Pilatus 300 K detector (Dectris) was used to collect thetwo-dimensional (2D) scattering patterns. Solutions were prepared bydiluting the NPs to ˜1 wt. % to avoid structure factor contributions. NPsolutions were measured within sealed glass capillaries. A blank sampleconsisting of a capillary with only toluene/TFT was measured under thesame conditions for background subtraction. SAXS data were acquired for30 minutes at room temperature with an X-ray flux of 21.4 M photons persecond incident upon the sample and a sample-to-detector distance of 425mm. Data were processed using SAXSGUI and custom MATLAB scripts. Thescattering form factor was fitted as a Gaussian number averagedistribution of hard spheres.

Vapor Swelling Chamber

The vapor chamber was built in-house using a bubbler mounted in a waterbath to maintain constant temperature. A dry air line with a flowcontroller was plumbed into the bubbler to generate vapor at a fixedrate of 27 mL/min. The same flow was also used as a purge line afterbypassing the bubbler. The output line was directed into a largetemperature-controlled oven set to 35° C. containing a long copper coilto equilibrate the vapor temperature before directing the gas phase intoa 0.2 L glass chamber housing the QCM crystal. The exhaust line wasplumbed from the glass chamber into a fume hood. Glass and metalconnectors were used as much as possible to eliminate diffusive uptakeof solvents into plastics.

Quartz Crystal Microbalance Measurements

Quartz crystals with 6 MHz resonance frequency were used. NP films werespin coated at 5,000 rpm from 1 wt % solutions onto QCM crystals. Thecrystal was then measured using a Colnatec Phoenix head and a ColnatecEon-LT monitor. RC cut QCM crystals were chosen to minimize temperatureeffects. Each measurement started with a system purge of dry air at thesame flow rate of 27 mL/min followed by vapor exposure until the filmreached equilibrium, ranging from 25-50 minutes. Following exposure toeach solvent, the crystal was again subjected to a purge to removephysiosorbed solvent and restore the baseline QCM frequency. The changesin QCM resonant frequency were recorded 5 times per second. Thefrequency decrease corresponding to mass uptake was found to equilibratewith a single exponential decay. All data were measured for at least 1.8times the fitted time constant (>83% progress towards equilibrium) toyield the equilibrium molecular uptake. The resulting frequency data wasanalyzed using custom MATLAB R2016b scripts. The changes to QCM resonantfrequency were converted to the corresponding mass change using theZ-Match method:

$\begin{matrix}{{\Delta \; m} = {{\frac{v_{q} \cdot \rho_{q}}{2\; {\pi \cdot z \cdot f_{1}}} \cdot {\tan^{- 1}\left( {z \cdot {\tan \left( \frac{\pi \cdot \left( {f_{0} - f_{1}} \right)}{f_{0}} \right)}} \right)}} = \frac{g}{{cm}^{2}}}} & (1)\end{matrix}$

where Δ_(m) is the change in mass (g/cm2), uq is the frequency constant(333,600 cm/sec), p_(q) is the density of quartz (2.648 gm/cm), Z is theZ-factor (1 for mass loadings less than 10-20% frequency shift), f₁ isthe final resonant frequency, and f₀ is the initial resonant frequency.The NP-film mass (g/cm2) was determined by using the resonant frequencyof the bare QCM as f0 and the resonant frequency with the NP-film as f₁.The molecular mass uptake (g/cm2) was determined similarly by using theresonant frequency of the NP-film as f0 and the resonant frequency withthe NP-film under saturated vapor as f₁.

Calculation of Solvent-NP Interaction

The molecule-NP interaction was compared for each solvent as a functionof the NP ligand shell. The molecular uptake for each film wasnormalized by the NP film mass to yield comparable relative extents ofuptake. The ratio of (g_(molecule)/cm²)/(g_(film)/cm²) yieldedg_(molecule)/g_(film). The vapor pressure was maintained constant foreach solvent since the molecular uptake mass (g_(molecule)) is dependentupon vapor pressure.

Ligand Surface Density Calculations

The ligand surface density for NPs was determined using a combination ofUV-Vis and quantitative ¹H NMR. The concentration of the gold NPs wasanalyzed using a Shimadzu UV-2450 Spectrometer over an absorbance rangeof 600 to 400 nm. Samples were prepared at approximately 0.2 mg of NPsper 1 mL solvent and placed in a fused quartz glass cuvette with a 1 cmpath length. The concentration was calculated using Lambert-Beer lawusing the absorbance at 508 nm and the extinction coefficient based onthe known NP diameter. The extinction coefficients were calculated usingthe constants k (3.32111) and a (10.80505).

The ligand concentration was quantified after iodine decomposition using¹NMR spectroscopy with an internal standard of known concentration. Herethe ratio of the internal standard (1,4-Difluorobenzene) to the ratio ofthe α H1 peaks of each ligand were used to quantify the concentration ofeach ligand as detailed elsewhere, see Smith, A. M.; Johnston, K. A.;Crawford, S. E.; Marbella, L. E.; Millstone, J. E. Ligand DensityQuantification on Colloidal Inorganic Nanoparticles. Analyst 2017, 142,11-29, which is hereby incorporated by reference. Iodine decompositionwas carried out by adding 1-3 mg of metallic iodine to the dispersedNPs, the particles were left to decompose for at least 12 hours toensure complete disulfide formation leading to a color change from deepred to clear violet. After twelve hours the black precipitate wasfiltered off and the solution was taken for 1H NMR analysis.

While the present subject matter has been described in detail withrespect to specific exemplary embodiments and methods thereof, it willbe appreciated that those skilled in the art, upon attaining anunderstanding of the foregoing may readily produce alterations to,variations of, and equivalents to such embodiments. Accordingly, thescope of the present disclosure is by way of example rather than by wayof limitation, and the subject disclosure does not preclude inclusion ofsuch modifications, variations and/or additions to the present subjectmatter as would be readily apparent to one of ordinary skill in the artusing the teachings disclosed herein.

What is claimed is:
 1. A quartz crystal microbalance method fordetecting molecular interactions with nanoparticles to quantifymolecule-nanoparticle interactions comprising: preparing a nanoparticlefilm on a quartz crystal; exposing the nanoparticle film to at least onemolecular vapor; and quantifying mass uptake via analyzing a resonantfrequency of the quartz crystal.
 2. The method of claim 1, furthercomprising correlating nonmonotonic uptake trends to ligand shellmorphologies as a function of confinement and cavitation effects.
 3. Themethod of claim 1, wherein non-solvents are used to quantifymolecular/nanoparticle interactions.
 4. The method of claim 1, whereindetermining morphology of at least one molecule/nanoparticle shell isaccomplished via analyzing nuclear magnetic resonance chemical shifts.5. The method of claim 4, wherein interfaces of different ligands have adifferent chemical shift wherein extent of chemical shift is a weightedaverage of at least one local ligand environment.
 6. The method of claim1, wherein molecular uptake is measured without requiring a solvationsphere.
 7. The method of claim 1, wherein the nanoparticle film isformed by spin coating.
 8. The method of claim 1, further comprisingcomparing molecular mass uptake to nanoparticle film mass to quantifyextent of uptake.
 9. The method of claim 1, further comprising probingfor non-monotonic trends in molecule-nanoparticle interactions withchanges to ligand composition.
 10. The method of claim 1, furthercomprising eliminating nanoparticle size distribution as a variable viaemploying ligand exchange.
 11. A method to quantify mixed ligand shellmolecule-nanoparticle interaction comprising: measuring vapor phaseuptake of molecules into a solid nanoparticle film, deposited on acrystal, via nuclear magnetic resonance, wherein the nanoparticle filmcomprises mixed ligand nanoparticles with constant size and variablecomposition; and wherein the method is independent of solvationcriteria.
 12. The method of claim 11, wherein the crystal is quartz. 13.The method of claim 11, wherein patchy ligand morphologies exhibit moremolecule uptake than either stripe-like or mono-ligand nanoparticles.14. The method of claim 11, wherein measurements occur without requiringa solvation shell.
 15. The method of claim 11, wherein ligand strippingis employed.
 16. The method of claim 11, comprising eliminatingnanoparticle size distribution as a variable via employing ligandexchange.